Fixing hippo neurons to assess endogenous NEMO during oxidative stress

We altered a previously developed method (dx.doi.org/10.17504/protocols.io.bxpfpmjn) in order to investigate the interaction of endogenous NEMO with damaged mitochondria in Hippocampal rat neurons. Mitochondrial damage was induced by the administration of 35 nM Antimycin A, a complex III inhibitor, over 1 hr. Here, we visualized neuronal mitochondria with a mito-targeted construct, Mito-SNAP, and a fluorescent SNAP ligand, JaneliaFluor 646, though other mitochondrial markers may be suitable. Endogenous NEMO was visualized with the commercially available anti-NEMO primary antibody (abcam). AntA-treated neurons exhibited fragmented mitochondria. We did not observe a change in the appearance of NEMO labeling in AntA treated cells versus non-treated cells.